Solid-phase peptide synthesis (SPPS) involves a covalent attachment step (i.e., anchoring) that links the nascent peptide chain to an insoluble polymeric support (i.e., support material) containing appropriate functional groups for attachment. Subsequently, the anchored peptide is extended by a series of addition (deprotection/coupling) cycles that involve adding Nxcex1-protected and side-chain-protected amino acids stepwise in the C to N direction. Once chain assembly has been accomplished, protecting groups are removed and the peptide is cleaved from the support.
Typically, SPPS begins by using a handle to attach the initial amino acid residue to the functionalized polymeric support. A handle (i.e., linker) is a bifunctional spacer that, on one end, incorporates features of a smoothly cleavable protecting group, and on the other end, a functional group, often a carboxyl group, that can be activated to allow coupling to the functionalized polymeric support. Known handles include acid-labile p-alkoxybenzyl (PAB) handles, photolabile o-nitrobenzyl ester handles, and handles such as those described by Albericio et al., J. Org. Chem., 55, 3730-3743 (1990) and references cited therein, and in U.S. Pat. No. 5,117,009 (Barany) and U.S. Pat. No. 5,196,566 (Barany et al.).
For example, if the support material is prepared with amino-functionalized monomers, typically, the appropriate handles are coupled quantitatively in a single step onto the amino-functionalized supports to provide a general starting point of well-defined structures for peptide chain assembly. The handle protecting group is removed and the C-terminal residue of the Nxcex1-protected first amino acid is coupled quantitatively to the handle. Once the handle is coupled to the solid-phase and the initial amino acid or peptide is attached to the handle, the general synthesis cycle proceeds. The synthesis cycle generally consists of deprotection of the Nxcex1-amino group of the amino acid or peptide on the resin, washing, and, if necessary, a neutralization step, followed by reaction with a carboxyl-activated form of the next Nxcex1-protected amino acid. The cycle is repeated to form the peptide or protein of interest. Solid-phase peptide synthesis methods using functionalized insoluble supports are well known. See, for example, Merrifield, J. Am. Chem. Soc., 85, 2149 (1963); Barany and Merrifield, In The Peptides, Vol. 2, pp. 1-284 (1979); Gross, E. and Meienhofer, J., Eds., Academic: New York; and Barany et al., Int. J. Peptide Protein Res., 30, 705-739 (1987).
Most current methods of SPPS rely on the a-carboxyl function of the eventual C-terminal amino acid residue to achieve anchoring to the support. However, this approach limits SPPS to the formation of peptides having acid, amide, or monosubstituted amide functionality, for example, as the C-terminal functionality, unless more complex procedures are used. Furthermore, certain functionalities, such as aldehydes, cannot typically be obtained using this approach. Cyclic peptides are also not possible using this method. Also, racemization of sensitive amino acid residues in the synthesis of peptide acids is a problem using this method.
Side-chain anchoring, i.e., methods of SPPS that use amino acids with side-chain functional groups for attachment of peptides, is potentially useful for the formation of unusual C-terminal functionalities as well as cyclic peptides. However, side-chain anchoring is inherently limited to certain trifunctional amino acids. Therefore, it would be desirable to develop a general method of SPPS that: (1) allows for the preparation of a wider variety of peptides; (2) does not typically result in racemization of sensitive amino acid residues; and (3) can incorporate a wider variety of amino acids into cyclic peptides.
The present invention provides a method of forming a support material linked to an amine-containing organic group for solid phase organic synthesis comprising:
(a) attaching a preformed divalent linker to a support material; and
(b) attaching an amine-containing organic group to the preformed divalent linker;
wherein steps (a) and (b) are carried out to form a support material linked to an amine-containing organic group of the following formula (Formula I): 
xe2x80x83wherein:
(i) Ŝ represents a support material;
(ii) L represents a divalent linker;
(iii) Y represents H or a protecting group; and
(iv) R1, R2, and R3 are each independently H or an organic group.
A preferred divalent linker (L) is of the formula (Formula II): 
wherein:
each U is independently selected from the group consisting of an alkyl group, an alkoxy group, an aryl group, an alkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthio group, an arylthio group, an alkylamido group, an alkylsulfinyl group, an alkylsulfonyl group, an alkylsulfoxide group, a halogeno group, and a nitro group, wherein any two U groups can be joined to form a ring; W is a functionalized spacer group for anchoring the linker to the support material; R5 and R6 are each independently H, an alkyl group, or an aryl group; and x=0-4.
To synthesize an organic compound, such as a peptide, once the support material of Formula I is prepared, a second organic group is attached to the N atom to build an organic compound. This is done using standard solid phase synthesis techniques and repeated addition cycles of deprotection and coupling.
The present invention also provides a method of synthesizing an organic compound comprising:
(a) providing an aldehyde-functionalized support material having the following formula (Formula III): 
xe2x80x83wherein:
(i) Ŝ represents a support material;
(ii) V is NH, S or O;
(iii) T is O, NH, NHC(O)R4, or S, wherein R4 is an alkylene group, an arylene group, or an aralkylene group;
(iv) R7 is an alkylene group, an arylene group, or an oxyalkylene group;
(v) each U is independently selected from the group consisting of an alkyl group, an alkoxy group, an aryl group, an alkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthio group, an arylthio group, an alkylamido group, an alkylsulfinyl group, an alkylsulfonyl group, an alkylsulfoxide group, a halogeno group, and a nitro group, wherein any two U groups can be joined to form a ring;
(vi) x=0-4; and
(vii) n=1-18;
(b) attaching an amine-containing organic group to the aldehyde functionality under reducing conditions; and
(c) attaching a second organic group to the N atom of the amine-containing organic group to build an organic compound.
The aldehyde-functionalized support material of Formula III is also provided, along with a kit for synthesizing an organic compound. The kit includes an aldehyde-functionalized support material having the Formula III and instructions for preparing an organic compound on the aldehyde-functionalized support material.
The present invention also provides a support material linked to an amine-containing organic group for solid-phase synthesis of an organic compound, wherein the support material has the following formula (Formula I): 
wherein:
(a) Ŝ represents a support material;
(b) L represents a divalent linker;
(c) Y represents H or a protecting group;
(d) R1 and R2 are each independently H or an organic group; and
(e) R3 is an organic group having a protecting group Z that is removable under mild conditions.
The present invention also provides a preformed linker having an amine-containing organic group attached thereto, of the formula (Formula IV): 
wherein:
(a) L represents a divalent linker;
(b) Q represents a group selected from the group consisting of C(O)OH, C(O)OPfp, C(O)F, C(O)Br, C(O)Cl, OH, Br, Cl;
(c) Y represents H or a protecting group;
(d) R1 and R2 are each independently H or an organic group; and
(e) R3 is an organic group having a protecting group Z that is removable under mild conditions.
As used herein, the term xe2x80x9corganic groupxe2x80x9d means a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., aralkyl groups). In the context of the present invention, the term xe2x80x9caliphatic groupxe2x80x9d means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example. The term xe2x80x9calkyl groupxe2x80x9d means a saturated linear or branched hydrocarbon group including, for example, methyl, ethyl, isopropyl, t-butyl, heptyl, dodecyl, octadecyl, amyl, 2-ethylhexyl, and the like. The term xe2x80x9calkenyl groupxe2x80x9d means an unsaturated, linear or branched hydrocarbon group with one or more carbon-carbon double bonds, such as a vinyl group. The term xe2x80x9calkynyl groupxe2x80x9d means an unsaturated, linear or branched hydrocarbon group with one or more carbon-carbon triple bonds. The term xe2x80x9ccyclic groupxe2x80x9d means a closed ring hydrocarbon group that is classified as an alicyclic group, aromatic group, or heterocyclic group. The term xe2x80x9calicyclic groupxe2x80x9d means a cyclic hydrocarbon group having properties resembling those of aliphatic groups. The term xe2x80x9caromatic groupxe2x80x9d or xe2x80x9caryl groupxe2x80x9d means a mono- or polycyclic aromatic hydrocarbon group. The term xe2x80x9cheterocyclic groupxe2x80x9d means a closed ring hydrocarbon in which one or more of the atoms in the ring is an element other than carbon (e.g., nitrogen, oxygen, sulfur, etc.).
As is well understood in this technical area, a large degree of substitution is not only tolerated, but is often advisable. Substitution is anticipated on the materials of the present invention. As a means of simplifying the discussion and recitation of certain terminology used throughout this application, the terms xe2x80x9cgroupxe2x80x9d and xe2x80x9cmoietyxe2x80x9d are used to differentiate between chemical species that allow for substitution or that may be substituted and those that do not allow or may not be so substituted. Thus, when the term xe2x80x9cgroupxe2x80x9d is used to describe a chemical substituent, the described chemical material includes the unsubstituted group and that group with O, N, or S atoms, for example, in the chain as well as carbonyl groups or other conventional substitution. Where the term xe2x80x9cmoietyxe2x80x9d is used to describe a chemical compound or substituent, only an unsubstituted chemical material is intended to be included. For example, the phrase xe2x80x9calkyl groupxe2x80x9d is intended to include not only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, t-butyl, and the like, but also alkyl substituents bearing further substituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus, xe2x80x9calkyl groupxe2x80x9d includes ether groups, haloalkyls, nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc. On the other hand, the phrase xe2x80x9calkyl moietyxe2x80x9d is limited to the inclusion of only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, t-butyl, and the like.
The present invention provides a method for preparing organic compounds, particularly peptides, using solid-phase synthesis. The method involves anchoring an amine-containing organic group, which is the starting point for building the organic compound, to a support material using a divalent linker. Specifically, if peptides are being prepared, the amino acid residue or peptide is anchored through its backbone to the support material. This is in contrast to conventional methods of solid-phase peptide synthesis, for example, that involve anchoring the amine-containing organic group through a side-chain functional group of an amino acid residue, or through the a-carboxyl functionality of the eventual C-terminal amino acid residue.
This backbone amide linker (BAL) approach for the preparation of peptides avoids some of the aforementioned problems and allows for the preparation of peptides or other organic compounds having a variety of C-terminal functionalities, e.g., not only acids, but also thioacids and thioesters, alcohols, disubstituted amides, and aldehydes, among others. It also allows for the preparation of cyclic peptides using a wider variety of amino acids. Also, this approach is advantageous because there is little racemization of sensitive amino acid residues at room temperature.
The support material linked to an amine-containing organic group has the following general formula (Formula I): 
wherein:
Ŝ represents a support material, typically a solid support, which may include a variety of functional groups for attachment, and may or may not include a spacer; L represents a divalent linker; Y represents H or a protecting group, such as an Nxcex1-amine protecting group; and R1, R2, and R3 are each independently H or an organic group.
The support material of Formula I is prepared by attaching a preformed divalent linker to a support material, and attaching an amine-containing organic group to the preformed divalent linker. These two steps can be carried out in either order. That is, the amine-containing organic group can be attached to the preformed divalent linker prior to attaching the preformed divalent linker to the support material. Alternatively, the preformed divalent linker can be attached to the support material prior to attaching the amine-containing organic group to the preformed divalent linker.
As used herein, a preformed divalent linker (i.e., handle) is one that is prepared and then added to the support material as opposed to being formed or built up on the support material. Although all support materials with linkers attached thereto described herein are not made using preformed handles, it is particularly desirable to do so. Thus, the methods of the present invention attach a preformed divalent linker to a support material either before or after it is attached to an amine-containing organic group.
Advantageously, in preferred embodiments, the method of preparing the support material of Formula I is carried out under relatively mild conditions. Preferably, the step of attaching the amine-containing organic group to the divalent linker, and more preferably both the step of attaching the amine-containing organic group to the divalent linker and the step of attaching the divalent linker to the support material, is carried out at a temperature of no greater than 35xc2x0 C. This reduces the chances of racemization of any chiral groups, such as chiral amino acid residues. The temperature at which either or both of these steps is carried out is more preferably about 0-30xc2x0 C., and most preferably about 20-25xc2x0 C. Typically, either or both of these steps is carried out for no greater than about 30 hours each, preferably for no greater than about 10 hours, more preferably for no greater than about 5 hours, and most preferably for no greater than about 2 hours each.
In the amine-containing organic group (i.e., xe2x80x94N(Y)xe2x80x94C(R1)(R2)(R3)), the groups R1, R2, and R3 are each independently H or an organic group. They can be a wide variety of organic groups, such as alkyls, aryls, heterocyclics, etc. Typically, at least one of R1, R2, and R3 is an amino acid side chain, which can be proteinogenic or non-proteinogenic amino acid side-chains.
Preferably, R1, R2, and R3 are each independently H or a (C1-C18)alkyl group, a (C6-C8)aryl group, a (C1-C18)alk(C6-C18)aryl group, a (C5-C18)heterocyclic group, or a (C1-C18)alk(C3-C18)heterocyclic group. More preferably, at least one of R1, R2, and R3 is selected from the group consisting of a xe2x80x94CH3, xe2x80x94CH(CH3)2, xe2x80x94CH(CH3)(CH2CH3), xe2x80x94CH2xe2x80x94CH(CH3)2, xe2x80x94(CH2)nX (n=1-4), and xe2x80x94CH(CH3)X group wherein X is selected from the group consisting of xe2x80x94OH, xe2x80x94OCH3, xe2x80x94NO2, xe2x80x94NH2, xe2x80x94SH, xe2x80x94SCH3, xe2x80x94C(O)OH, xe2x80x94C(O)NH2, xe2x80x94C6H5, xe2x80x94C6H4OH, indoyl, imidazoyl, and protected derivatives thereof.
For certain embodiments of the support material of Formula I, R1 and R2 are each independently H or an organic group as defined above, and R3 is an organic group having a protecting group Z that is removable under mild conditions (e.g., moderate or weak acid, moderate or weak base, photolysis, thiolysis, palladium-catalyzed or rhodium-catalyzed nucleophilic transfer, hydrogenation, or fluoridolysis). As used herein, moderate or weak acids are those with an HO of xe2x88x925 or higher, as defined by J. P. Tam et al., in The Peptides, Vol. 9 (S. Udenfriend and J. Meienhofer, Eds.), pp. 185-248, Academic Press, New York (1987). Examples of such acids include, but are not limited to, hydrochloric, acetic, dilute sulfuric, and trifluoroacetic acid. Moderate or weak bases are those having conjugate acids with a pka of of 15.0 or less. Examples of such bases include, but are not limited to, piperdine, morpholine, and 1,8-diazabicyclo[5.4.0]undec-7-ene.
Suitable protecting groups Z depend on the functionality of R3, which could include amines, carbonyls, hydroxyls, carboxylic acids, aldehydes, thiocarbonyls, etc. In preferred embodiments of the support material of Formula I, R3 is selected from the group consisting of xe2x80x94C(O)OZ, xe2x80x94C(O)SZ, xe2x80x94C(S)OZ, xe2x80x94C(OZ)2R4 (wherein R4 is an alkyl group, an aryl group, or an aralkyl group), and xe2x80x94C(OZ)2H. Examples of suitable protecting groups Z include allyl, (C1-C4)alkyls, trityl, etc. Preferably, the protecting group Z is selected from the group consisting of methyl, t-butyl, and allyl moieties.
The protecting group Y of the support material of Formula I can be a wide variety of protecting groups that can be removed using conditions that do not cleave the Nxe2x80x94L bond, do not remove the linker from the support material, and do not adversely affect the compound (e.g., the peptide) being formed on the support material. Thus, the linker L and the protecting group Y are preferably chosen such that they can be removed in an orthogonal fashion. An orthoganol protection scheme is defined as one which makes use of two or more independent classes of groups, each one removed through a different chemical mechanism, allowing them to be removed in any order and in the presence of all the other classes.
Suitable protecting groups (Y) include, for example, those that can be removed using a wide variety of known conditions. Preferably, the protecting group Y is chosen such that it can be removed using mild conditions such as moderate or weak acid, moderate or weak base, photolysis, thiolysis, palladium- or rhodium-catalyzed nucleophilic transfer, hydrogenation, and fluoridolysis.
Examples of suitable protecting groups (Y) include, but are not limited to: formyl; alkyl groups; aryl groups such as p-phenylbenzyl and 9-phenylfluorenyl; alkenyl groups; aralkyl groups; aralkenyl groups; alkylcarbonyl groups such as acetyl (Ac) and derivatives of acetyl such as acetoacetyl, mono-, di-, and tri-halogen substituted acetyl (e.g., chloroacetyl, trichloroacetyl, trifluoroacetyl, etc.), o-nitrophenoxyacetyl, and phenylacetyl; arylcarbonyl groups such as benzoyl and p-nitrobenzoyl; alkyloxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, isobutoxycarbonyl, 1-adamantyloxycarbonyl, 1,1-dimethyl-2-cyanoethoxycarbonyl, 1,1-dimethyl-2,2-dibromoethoxycarbonyl, 1,1-dimethyl-2,2,2-trichloroethoxycarbonyl, diisopropylmethoxycarbonyl, 2-iodoethoxycarbonyl, and 2-(trimethylsilyl)ethoxycarbonyl; aryloxycarbonyl groups such as m-nitrophenyloxycarbonyl and phenyloxycarbonyl; alkenylmethoxycarbonyl groups such as allyloxycarbonyl and 4-nitrocinnamyloxycarbonyl; alkenyloxycarbonyl groups such as vinyloxycarbonyl; aralkyloxycarbonyl groups such as 1-methyl-1-phenylethoxycarbonyl, 1-methyl-1-(4-pyridyl)ethoxycarbonyl, di(2-pyridyl)methoxycarbonyl, 1-methyl-1-(4-biphenyl)ethoxycarbonyl, and 9-fluorenylmethoxycarbonyl; cycloalkyloxycarbonyl groups such as cyclobutyloxycarbonyl, cyclohexyloxycarbonyl, cyclopentyloxycarbonyl, and 1-methyl-1-cyclohexyloxycarbonyl; alkylaminooxycarbonyl groups such as N-hydroxypiperidinyloxycarbonyl; sulfenyl groups such as o-nitrophenylsulfenyl and 3-nitropyridinesulfenyl; and sulfonyl groups such as p-toluenesulfonyl and xcex2-(trimethylsilyl)ethanesulfonyl.
The protecting group Y is preferably an Nxcex1-amine protecting group. Examples of suitable Nxcex1-amine protecting groups include, for example, Fmoc, Aloc, Boc, Ddz, Npys, Nvoc, Bpoc, Teoc, Trt, SES, allyl, and t-Bu. These abbreviations are defined in the Examples section below. A preferred group of Nxcex1-amine protecting groups include, Fmoc, Aloc, and Boc.
The linker L in the support material of Formula I can be a wide variety of handles used in solid phase peptide synthesis. The linker L is a bifunctional spacer that, on one end, incorporates features of a smoothly cleavable protecting group, and on the other end, a functional group, often a carboxyl group, that can be activated to allow coupling to the functionalized support material. The linker can be a preformed linker or handle or it can be prepared on the support material. Suitable linker (L) examples include the PAL handle [5-(4xe2x80x2-aminomethyl-3xe2x80x2,5xe2x80x2-dimethoxyphenoxy)valeric acid] and the XAL handle [5-(9-aminoxanthen-2-oxy)valeric acid]. Other suitable linkers include handles such as 4-(xcex1-aminobenzyl)phenoxyacetic acid, 4-(xcex1-amino-4xe2x80x2-methoxybenzyl)phenoxybutyric acid, 4-(xcex1-amino-4xe2x80x2-methoxybenzyl)-2-methylphenoxyacetic acid, 2-hydroxyethylsulfonylacetic acid, 2-(4-carboxyphenylsulfonyl)ethanol, and those disclosed in G. B. Fields et al., Synthetic Peptides: A User""s Guide, 1990, 77-183, G. A. Grant, Ed., W. H. Freeman and Co., New York.
A preferred group of linkers is of the following formula (Formula II): 
wherein:
each U is independently selected from the group consisting of an alkyl group, an alkoxy group, an aryl group, an alkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthio group, an arylthio group, an alkylamido group, an alkylsulfinyl group, an alkylsulfonyl group, an alkylsulfoxide group, a halogeno group, and a nitro group, wherein any two U groups can be joined to form a ring; W is a functionalized spacer group for anchoring the linker to the support material; R5 and R6 are each independently H, an alkyl group, or an aryl group; and x=0-4.
Preferably, W is selected from the group consisting of xe2x80x94(CH2)nC(O)NHxe2x80x94, xe2x80x94O(CH2)nC(O)NHxe2x80x94, xe2x80x94NH(CH2)nC(O)NHxe2x80x94, xe2x80x94OC(O)(CH2)nC(O)NHxe2x80x94, xe2x80x94C(O)(CH2)nC(O)NHxe2x80x94, xe2x80x94C(O)O(CH2)nC(O)NHxe2x80x94, xe2x80x94NHC(O)(CH2)nC(O)NHxe2x80x94, xe2x80x94O(CH2)C6H4C(O)NHxe2x80x94, xe2x80x94C(O)O(CH2)C6H4C(O)NHxe2x80x94, xe2x80x94OC(O)C6H4C(O)NHxe2x80x94, xe2x80x94OC(O)(CH2CH2O)nC(O)NHxe2x80x94, xe2x80x94O(CH2CH2O)nC(O)NHxe2x80x94, xe2x80x94NH(CH2CH2O)nC(O)NHxe2x80x94, and xe2x80x94NHC(O)(CH2CH2O)nC(O)NHxe2x80x94 wherein n=1-18.
The present invention also provides a useful method of synthesizing an organic compound using an aldehyde-functionalized support material. This aldehyde-functionalized support material has the following formula (Formula III): 
wherein:
Ŝ represents a support material; V is NH, S or O; T is O, NH, NHC(O)R4, or S, wherein R4 is is an alkylene group, an arylene group, or an aralkylene group; R7 is an alkylene group, an arylene group, or an oxyalkylene group; each U is independently selected from the group consisting of an alkyl group, an alkoxy group, an aryl group, an alkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthio group, an arylthio group, an alkylamido group, an alkylsulfinyl group, an alkylsulfonyl group, an alkylsulfoxide group, a halogeno group, and a nitro group, wherein any two U groups can be joined to form a ring; x=0-4; and n=1-18. Preferably, R1 is an alkylene group and n=1-4.
An amine-containing organic group is then typically attached to the aldehyde functionality under reducing conditions. Typically, this is carried out at a temperature of no greater than 35xc2x0 C. for no greater than about 10 hours. As used herein, reducing conditions include NaBH3CN, NABH4, cat.H2, and NaBH(OAc)3. A second organic group, which may be a protected amino acid, is then attached to the N atom of the amine-containing group to build an organic compound. This is typically carried out in a nonprotic solvent selected from the group consisting of CH2Cl2, ClCH2CH2Cl, tetrahydrofuran, CH3CN, toluene, pyridine, dioxane, diethyl ether, and benzene. The organic compounds prepared may or may not be a peptide, which may or may not be cyclic, or have a broad range of functional groups on the peptide being formed. For example, the method of the present invention can provide a terminal carboxylic acid group, ester group, aldehyde group, thioacid group, and thioester group.
The present invention also provides a kit that includes the aldehyde-functionalized support material of Formula III and instructions for preparing an organic compound on the aldehyde-functionalized support material.
The present invention also provides a preformed linker having an amine-containing organic group attached thereto, of the formula (Formula IV): 
wherein:
L represents a divalent linker; Q represents a group selected from the group consisting of C(O)OH, C(O)OPfp, C(O)F, C(O)Br, C(O)Cl, OH, Br, Cl; Y represents H or a protecting group; R1 and R2 are each independently H or an organic group; and R3 is an organic group having a protecting group Z that is removable under mild conditions.
The linker is attached to functionalized support materials or to spacer arms attached to the support materials. Typically, the support material includes hydroxyl, carboxyl, or amino functional groups, although other functional groups such as thiol, halogens, and silyl are possible. The support material can also include a spacer. Typically, a spacer is an alkyl chain (preferably a (C1-C20)alkyl chain) substituted with an amino group and a carboxyl group.
A variety of functionalized support materials can be used. They can be of inorganic or organic materials and can be in a variety of forms (e.g., membranes, particles, spherical beads, fibers, gels, glasses, etc.). Examples include, porous glass, silica, polystyrene, polydimethylacrylamides, cotton, paper, and the like. Examples of suitable support materials are described by G. B. Fields et al., Int. J. Peptide Protein Res., 1990, 35, 161-214 and G. B. Fields et al., Synthetic Peptides: A User""s Guide, 1990, 77-183, G. A. Grant, Ed., W. H. Freeman and Co., New York. Functionalized polystyrene, such as amino-functionalized polystyrene, aminomethyl polystyrene, aminoacyl polystyrene, p-methylbenzhydrylamine polystyrene, or polyethylene glycol-polystyrene resins can be used for this purpose. Polyethylene glycol-polystyrene (PEG-PS) graft copolymers functionalized with amino groups are particularly useful support materials. Suitable PEG-PS resins are available from PerSeptive BioSystems (Framingham, Mass.) and are described in U.S. Pat. No. 5,235,028 (Barany et al.).
The support materials of the present invention can be prepared by attaching the linker to the support material and then attaching the amine-containing organic group, e.g., amino acid or peptide. The attachment reaction of the linker to the support material can be carried out using standard coupling methods, e.g., acylations or alkylations, as disclosed in, for example, F. Albericio et al., J. Org. Chem., 1990, 55, 3730-3743, or alkylations. For example, acylations promoted by N,Nxe2x80x2-dicyclohexylcarbodiimide (DCC), or N,Nxe2x80x2-diisopropylcarbodiimide (DIPCDI) plus 1-hydroxybenzotriazole (HOBt), or benzotriazoylyl-N-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) plus 1-hydroxybenzotriazole (HOBt). Typically, one equivalent of the linker is used for each equivalent of functional group, e.g., amino group, present on the support. After attaching the amine-containing group to the linker, the protecting group can be added.
Alternatively, the linker, amino acid, and protecting group (if used) can be combined to form an optionally protected amino acid preformed linker, which is then attached to the support material. For example, in one embodiment of the present invention, an aldehyde precursor of the PAL handle [5-(4xe2x80x2-aminomethyl-3xe2x80x2,5xe2x80x2-dimethoxyphenoxy)valeric acid], which is disclosed in Albericio and Barany, Int. J. Pept. Protein Res., 1987, 30, 206-216, can be coupled through a reductive amination procedure to the xcex1-amine of the prospective C-terminal amino acid or other amine-containing compound, which can be protected as a tert-butyl, methyl, trityl, or allyl ester, or modified to a dimethyl acetal. The resultant intermediates, all secondary amines, can be treated with Fmoc-Cl or Fmoc-OSu to give the corresponding protected amino acid (or other amine-containing compound) preformed handles in 40-70% yields.